The overall goal of the proposed work is to bridge the gap between biochemical rate constants measured in[unreadable] vitro and physiological function. The means by which this gap can be bridged is a computational model that[unreadable] recapitulates IKB/NF-KB signaling and genetic complementation to study the behavior of in vitro[unreadable] characterized mutants. Our goal is to relate biochemical and biophysical[unreadable] findings by co-PI's laboratories to physiologically relevant signal transduction characteristics, and to motivate[unreadable] their work by providing context and focus. The specific aims are: (1) Little is known about the degradation[unreadable] mechanism of free (not NF-KB-bound) IKB protein despite its enormous effect on the signaling characteristics[unreadable] of the pathway. We will use an unbiased proteomic approach to characterize the free IKB polypeptide and[unreadable] potential degradation intermediates. These studies will provide context and inform subsequent work. (2)[unreadable] Mutant IKBa proteins previously characterized in vitro, will be examined in vivo. These studies will correlate[unreadable] dynamic compact foldedness, thermal stability, proteasome sensitivity and in vivo degradation rates, thereby[unreadable] revealing which characteristics are important in IKBa half-life control. (3) Such mutants will be used to[unreadable] study the functional role of interaction and degradation rate constants in signaling. To that end we will[unreadable] construct a lentiviral complementation system that allows for faithful expression in IKB knockout cells.[unreadable] These studies will be guided by computational simulations to investigate the signal transduction[unreadable] characteristics of the IKB/NF-KB signaling module. (4) IKBa is thought to mediate post-induction repression[unreadable] of gene expression by removing NF-KB from promoter DNA. Regulated folding of sequences in the p65[unreadable] protein appear to play a role in vitro. Using knockout cell lines, complemented with appropriate mutants, we[unreadable] will investigate this process guided by in vitro studies. These studies will culminate with complementation of[unreadable] p65 knockout mice by lentiviral transgenesis.